We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 A. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.

We have used time-resolved phosphorescence anisotropy (TPA) to probe rotational dynamics of the rabbit skeletal sarcoplasmic reticulum Ca-ATPase (SERCA), to test the hypothesis, generated from X-ray crystallography, that large-scale structural changes are induced by Ca in this system. Previous TPA studies on SERCA used primarily erythrosin 5'-isothiocyanate (ErITC), which binds to the nucleotide-binding domain and inactivates the enzyme. To investigate rotational dynamics of the active enzyme, we labeled SERCA with erythrosin 5'-iodoacetamide, which binds to the phosphorylation domain and has a minimal effect on the calcium-dependent ATPase activity. In the absence of nucleotide and the presence of calcium, TPA results were similar to those observed previously with ErITC, consistent with the global uniaxial rotation of SERCA monomers and oligomers and small amplitude internal protein dynamics. The removal of Ca had only a slight effect, while the addition of adenosine 5'-triphosphate (ATP) increased the amplitude of internal dynamics and changed the probe's orientation, corresponding to tilting of the phosphorylation domain by at least 20 degrees . Ca partially reversed the ATP effects. A nonhydrolyzable ATP analogue had the same effects as ATP, showing that the observed changes were not dependent on active ion transport. Computational analysis indicates that these ligands affect primarily the internal dynamics of the enzyme, with negligible effects on global dynamics and enzyme association. Melittin, which has been shown to aggregate and inhibit SERCA, eliminated the nucleotide-induced internal dynamics and increased the final anisotropy. We propose that (i) the large Ca-dependent structural changes suggested by SERCA crystallography are more dependent on ATP than on Ca and (ii) aggregation-induced inhibition of SERCA is due to the functional coupling between global and internal protein dynamics.

Radiationless energy transfer between large molecules has been studied in samples in which a coulombic interaction can be expected to dominate other processes. Measurement of the effect of energy transfer on the lifetime of the donor yields information on the distance dependence of the interaction. The "Forster" formulation of energy transfer due to a coulombic dipole-dipole interaction has been found to describe accurately the results of both singletsinglet and triplet-singlet transfer. In the latter case, the experimental data are sufficiently precise to be analyzed in a manner suggested by lnokuti and Hirayama, leading to the conclusion that a pure dipole-dipole interaction induces the transfer.